The human embryonic stem cell (hESC), derived from the inner cell mass (ICM), offers both a model system for human development and a potentially unlimited source of graft material for cell-based therapies. The pluripotence of hESCs implies such cells'tremendous potential for tissue and function restoration, whereas it has vacated a practical approach to generate a large supply of uniform replacement cells from hESCs for treating diseases. Controlled differentiation of hESCs effectively into functional lineages has been one of the daunting challenges for fulfilling the therapeutic promise of hESCs and requires a through understanding of the molecular and cellular cues that direct hESC differentiation programs. Previous hESC differentiation procedures largely rely on the formation of multi-lineage aggregates where only a small fraction pursues a given phenotype, in part because it has been assumed that tissue and organ systems arise from the three embryonic germ layers. However, the nervous system and the heart are among the first tissue and organ systems formed from the cells of the ICM. It is deducible that the specification of early embryonic neural and cardiac lineages may occur directly from pluripotent hESCs. Therefore, I hypothesize that a minimal essential culture system will render signal molecules sufficient to specify pluripotent hESCs differentiate directly and exclusively into neuroectoderm and cardiomesoderm from where the nervous system and the heart evolve, respectively. This project will focus on uncovering hESC neural differentiation programs. This proposal allows a novel approach for direct induction of pluripotent hESCs exclusively into a rich collection of neural-restricted progenies for cell-based therapies as well as the development of an effective in vitro model system to investigate molecular controls in human embryonic neurogenesis. The feasibility of this project is supported by our preliminary data. These studies will tremendously improve our ability to manipulate hESC differentiation and help develop strategies for preventing and treating diseases. The breakthrough brought by this project will have significant impact on biomedical sciences. Public Health Relevance: This proposed research will reveal the biological pathways and molecular targets that control the formation of particular somatic phenotypes in human development, thereby aid the formulation of more efficient routes to derive an optimal source of somatic stem cells with neuronal potential from human embryonic stem cells (hESCs) for cell-based therapies.